1. Field of the Invention
This invention relates to the human anhidrotic ectodermal dysplasia (EDA) gene code for anhidrotic ectodermal dysplasia, an X-chromosomal recessive disorder. More particularly, the invention relates to various yeast artificial chromosomes (YACs) which contain all or a portion of the human EDA gene and to methods for making the same.
2. Description of the Prior Art
Physical maps of large chromosomal regions are defined by a series of DNA markers, preferably at closely and evenly distributed intervals. Such maps can be developed without cloning most of the chromosomal DNA, but it is advantageous to clone DNA in order to identify genes and study gene expression. Therefore, physical maps are preferably produced by reassembling chromosome equivalents from purified DNA.
The DNA molecule that makes up the X chromosome is much too large to be handled intact, so it must be broken into cloned fragments that are arranged by overlaps to create a contiguous map of the DNA. Larger clones, such as yeast artificial chromosomes (YACs), make physical mapping easier.
Two methods may be used to organize DNA fragments into a mapped region. In the first method, chromosome-specific probes are used to screen YAC libraries for cognate clones. Many such probes have been characterized and genetically or cytogenetically assigned to regions of the X chromosome. Probes defining genetically mapped, polymorphic loci are used to find corresponding larger YAC clones and provide markers that format the physical map. Such probes may be obtained as clones (in plasmids, phage, or cosmids) derived from flow-sorted chromosomes or genomic libraries constructed from somatic cell hybrids; by polymerase chain reaction (PCR)-based amplification of microdissected fragments of individual chromosomes; or by amplification of segments flanked by human-specific interspersed, repetitive sequences present in hybrid cells and YACs.
In the second method, clones for all or part of a chromosome are systematically analyzed by fingerprinting techniques, such as sizing restriction fragments or studying fragments that contain certain repetitive sequences. Overlaps between clones are then detected by computer analysis.
Two types of YAC libraries can be used to build an X physical map: total genomic libraries or X chromosome-specific libraries constructed from appropriate somatic hybrids. Chromosome-specific libraries have a smaller number of clones, and so favor screening with probes.
Because of the functional hemizygosity of the X chromosome, many translocations between X and an autosome as well as other structural abnormalities (such as deletions, duplications, and isochromosomes) are detected clinically. By means of the selectable markers described above, such rearranged chromosomes have been isolated in somatic cell hybrids and have provided a rich resource for interval mapping, especially in the pericentromeric region and in the middle and distal long arm regions.
At least 16 X-linked diseases have been cloned on the basis of prior knowledge of a defective protein. However, for most of the remaining diseases, the bio-chemical defect is unknown or very uncertain and it is necessary to use mapping strategies to identify the genes corresponding to the diseases.
Fortunately, X-linked diseases have features that facilitate positional cloning. Chromosomal assignment, which can be difficult to determine for rare autosomal diseases, is obvious for X-linked diseases because of the inheritance pattern. Rare affected females with balanced X-autosome translocations have been found for about 10 diseases. In these patients, the normal X chromosome is generally inactive, and the translocated X active, because of a selection process operating in early embryogenesis against cells carrying an inactive translocated X. Translocations that have a breakpoint within a gene will lead to expression of the corresponding disease, as the uninterrupted copy on the normal X is inactive. Such translocations have provided precise localization for the relevant disease genes that could be confirmed by linkage analysis in affected families and have been instrumental in the cloning of several genes (Mandel et al. 1992).
Anhidrotic (hypohidrotic) ectodermal dysplasia (EDA; Christ-Siemens-Touraine syndrome; CST syndrome; MIM 305100, McKusick 1990) is an X-linked recessive disorder linked with the absence or hypoplasia of hair, teeth, and sweat glands as main manifestations (Reed et al. 1970, Clarke 1987). Prenatal diagnosis of X-linked anhidrotic ectodermal dysplasia (EDA) was previously performed by the direct histological analysis of fetal skin obtained by late second trimester fetoscopy (Zonana et al. 1990). Zonana et al. report that recent gene mapping of the locus for the EDA gene to the region of Xq11-21.1 permits indirect prenatal diagnosis of the disorder by the method of linkage analysis, based on closely linked marker loci, during the first trimester of pregnancy.
The EDA gene has been mapped to Xq12-q13 by genetic linkage analysis using restriction fragment length polymorphisms (RFLP) markers (MacDermot et al. 1986, Kolvraa et al. 1986, Clarke et al. 1987, Hanauer et al. 1988, Zonana et al. 1988a). However, Goodship et al. report a family with anhidrotic ectodermal dysplasia in which the disease did not segregate with the Xq11-q13 region of the X chromosome, as expected (Goodship et al. 1990). Physical and linkage maps for the pericentromeric region of the X chromosome have been refined (Lafreniere et al. 1991, Jones et al. 1991), but the region in which the EDA gene resides has not previously been cloned.
A similar syndrome with anhidrosis and absence of sweat glands is known in the mouse, in which the mutant gene is called Tabby (Ta) (Blecher 1986). Consistent with the map position in man, the Ta gene has been mapped in syntenically corresponding region in the X chromosome of mouse (Brockdorff et al. 1991). Blecher et al. report that epidermal growth factor (EGF) induces development of dermal ridges and functional sweat glands in Ta/Y hemizygotes, indicating a role in mammalian morphogenesis and possible treatment of anhidrotic ectodermal dysplasia (Blecher et al., 1990).
Zonana et al. have defined the human DXS732 locus by a conserved mouse probe pcos169E/4 (DXCrc169 locus) that co-segregates with the mouse tabby (Ta) locus, a potential homologue to the EDA locus. Zonana et al. report that the absence of recombination between EDA and the DXS732 locus supports the hypothesis that the DXCrc169 locus in the mouse and the DXS732 locus in humans may contain candidate sequences for the Ta and EDA genes, respectively (Zonana et al. 1992)
Zonana et al. further disclose the genetic nature of this anhidrotic ectodermal dysplasia. More particularly, they have screened a panel of genomic DNA samples from 80 unrelated males with EDA and identified a single individual partially deleted at the DXS732 locus. The individual has the classical physical signs and symptoms of EDA, has no other phenotypic abnormalities, and does not have a cytogenetically detectable deletion in the Xq12-q13.1 region. Zonana et al. conclude that since the DXS732 locus contains a highly conserved sequence in both the mouse and the hamster, it must be considered as a candidate locus for the EDA gene (Zonana et al. 1993).
Thomas et al. disclose two female patients who express the full clinical spectrum of anhidrotic ectodermal dysplasia in association with different X-chromosome cyotogenetic rearrangements. Both patients have cytogenetic breakpoints within the Xq13.1 region. A probe derived from cell lines from the two patients was used to screen a panel of unrelated affected EDA males and identified a patient with an interstitial deletion (Thomas et al. 1993).
While none of the prior art have succeeded in isolating the EDA gene, cloning of the region in which the EDA gene resides would have practical benefits. These would include both the development of tests for clinical variants in the gene and applications to the study and control of optimal hair, tooth, skin, and sweat gland development, all of which require a functional EDA gene.
Therefore, it is desirable to further limit the segment of DNA that contains the human EDA gene and to provide various yeast artificial chromosomes (YACs) which contain all or a portion of the human EDA gene and specific probes for the human EDA gene sequences.